Background and purpose The origin of migraine pain is still elusive, but increasingly researchers focus on the neuropeptides in the perivascular space of cranial vessels as important mediators of nociceptive input during migraine attacks. The parasympathetic neurotransmitters, pituitary adenylate cyclase activating peptide-38 (PACAP38) and vasoactive intestinal peptide (VIP) may be released from parasympathetic fibres and activate sensory nerve fibres during migraine attacks. Triptans are effective and well tolerated in acute migraine management but the exact mechanism of action is still debated. Triptans might reduce circulating neuropeptides. To examine this question, we examined the effect of sumatriptan on VIP and PACAP levels in vivo, under conditions without trigeminovascular system activation. Methods In 16 healthy volunteers we measured VIP and PACAP levels before and after administration of subcutaneous sumatriptan. We simultaneously collected blood samples from the internal and external jugular, the cubital veins and the radial artery, thereby covering both the cerebral and systemic circulation. VIP and PACAP determinations were assayed blindly with respect to timing and vascular compartments, but with all samples of a patient in the same assay, to minimize the influence of interassay variation. Results We found no difference in VIP and PACAP concentrations between the internal and external jugular, the cubital veins and the radial artery, (P>0.05), and the circulating levels of VIP and PACAP did not change over time (P>0.05). We found excellent agreement between neuropeptide levels in the internal and the external jugular system. Conclusion Sumatriptan did not change the levels of circulating VIP and PACAP in the intra or extra cerebral circulation in healthy volunteers. Under baseline conditions, without trigeminovascular activation, sumatriptan does not affect the release of neuropeptides VIP and PACAP. Implications Our results indicate no effect of 5-HT1B/D receptor activation on circulating levels of VIP and PACAP in humans without trigeminovascular activation. Given that neuropeptides play an important role for migraine it would be interesting to conduct a similar study in a migraine population.